During the past twenty years, two methods have become established in the mass spectrometry of biological macromolecules: ionization by matrix-assisted laser desorption (MALDI), and electrospray ionization (ESI). The biological macromolecules to be analyzed are termed analyte molecules below. In the MALDI method, the analyte molecules are generally prepared on the surface of a sample support in a solid, polycrystalline matrix layer, and are predominantly ionized with a single charge, whereas in the ESI method they are dissolved in a liquid and are ionized with multiple charges. The two methods have made it possible to conduct mass-spectrometric analysis of biological macromolecules for genomic, proteomic and metabolomic investigations; their inventors, John B. Fenn and Koichi Tanaka, were awarded the Nobel Prize for chemistry in 2002.
In a MALDI sample preparation, there are 103 to 105 times as many matrix molecules as analyte molecules, and the matrix molecules form a polycrystalline layer in which the analyte molecules are integrated in the interior of the crystals or at their grain boundaries, largely without coming into contact with other analyte molecules. The matrix substance is selected in such a way that its molecules can absorb the ultraviolet light of the laser pulse, on the one hand, and can protonate the analyte molecules on the other. In the prior art, a small illumination area (called “spot” in the following), with a diameter of around 50 to 200 micrometers, on the prepared MALDI sample is briefly irradiated with a laser pulse that is strongly absorbed by the matrix molecules. The pulsed irradiation converts surface matrix material from the solid state into the plasma phase in only a few nanoseconds, during which many matrix molecules (or their fragments) are thermally ionized. The analyte molecules are usually ionized by being protonated or deprotonated in reactions with matrix molecules or matrix ions in the dense plasma. The plasma cloud expands into the vacuum in a few hundred nanoseconds, while all the molecules are continuously accelerated by friction in the expanding plasma, and undergoes strong adiabatic cooling in the expanding process. At some point in time during the expansion, the gas molecules cease to be in contact with each other: thereafter the ionization state of the molecules in the plasma cloud is frozen. The degree of ionization of the analyte molecules in conventional MALDI is reported to amount to only around 104. The analyte ions are predominantly singly charged. This process is a “soft ionization” because the analyte molecules are ionized as molecular ions without suffering breaking of bonds.
The ionization of the analyte molecules by the matrix is a function of the energy density in the laser spot, and this function is extremely nonlinear (according to several concurring literature references, the degree of ionization increases with the sixth to seventh power of the energy density). The first analyte ions appear at an energy density threshold of around 10 millijoules per square centimeter. At around 100 millijoules per square centimeter, at least a million times more ions are created; but this energy density constitutes an upper limit for a soft ionization, beyond which spontaneous fragmentations of the analyte molecules occur. The setting of the optimum energy density is critical because, on the one hand, the mass resolution of the time-of-flight mass spectrometer depends on the energy density and, on the other hand, only a maximum of around a thousand analyte ions may be produced per laser pulse. Otherwise the saturation limit of the ion detector system, which is usually equipped with an 8-bit DAC, is exceeded. For the analyses, it is important that individual ions can also be detected with certainty; with a maximum of a thousand ions in the strongest ion signal and a measuring rate of four gigahertz, the ions of the strongest ion signal are distributed over several measuring intervals in such a way that saturation is just avoided, but individual ions still generate detectable signals. Since a doubling of the energy density increases the degree of ionization of the analyte molecules by at least a factor of 26=64, this optimum energy density is only a factor of about 2.5 to 3.0 above the energy density threshold where the first ions appear. Total energy and energy density must be kept constant to about one percent; this keeps fluctuations in ion generation and in the degree of ionization at around 6 to 7 percent. A further increase in the energy density by a factor of 3 would be able to increase the degree of ionization for analyte molecules to more than 10 percent, but would hopelessly oversaturate the ion detector system. Furthermore, increasing the energy density causes a simultaneous increase in the number of “metastable” ions; these are ions that decay on their way to the ion detector and cannot reach the ion detector for ion-optical reasons. If the number of metastable ions becomes too high, the degree of ionization can still increase, but the number of detectable ions cannot.
The MALDI process is complex, and is affected by numerous factors, some of which are interdependent. Since the MALDI method was first published in 1988, many parameters have been investigated and varied. In spite of this, the processes in the matrix and in the vaporization cloud which lead to the ionization of the analyte molecules are still not completely understood and remain the subject of intensive research, see for example the paper by K. Dreisewerd, Chem. Rev. 103 (2003), 395-425: entitled “The Desorption Process in MALDI”.
The chemical parameters of the MALDI process, for example the type of matrix substances, the concentration ratio between matrix and analyte molecules, and the preparation conditions, have been thoroughly researched. For analyte molecules of different chemical substance classes, such as proteins or nucleic acids, over a hundred different chemical matrix substances have been elucidated, such as sinapic acid, DHB (2,5-dihydroxybenzoic acid), CHCA (α-cyano-4-hydroxycinnamic acid) or HPA (3-hydroxypicolinic acid), which affect the MALDI process in different ways and can be used for different purposes. The matrix substances are usually aromatic acids; the aromatic ring gives them a strong absorption capacity in the wavelength range between 330 and 360 nanometers, and as acids they can easily donate a proton.
A MALDI sample can be prepared in a number of different ways, for example with “dried droplet” preparation, or the more preferable thin-layer preparation. In “dried droplet” preparation, the matrix substance is dissolved together with the analyte molecules in a solvent before being applied to a sample support and then dried. This preparation distributes analyte molecules extremely inhomogeneously in the matrix crystal complexes, however, and can scarcely be used for quantitative analyses. With thin-layer preparation, on the other hand, the matrix substance is applied to the sample support without analyte molecules and is dried to give a thin polycrystalline matrix layer only a few micrometers thick. This thin matrix layer has a high absorptivity for peptides and proteins. A drop of an aqueous solution containing analyte molecules is then applied to the thin matrix layer; the drop spreads quickly over the whole thin layer, and the analyte molecules are uniformly absorbed. The water can even be removed. After final drying, special measures can be applied to partially redissolve the matrix layer, and the analyte molecules can be embedded uniformly in the matrix layer during the subsequent drying.
As far as the physical parameters of the MALDI process are concerned, investigations have so far chiefly focused on examining how ionization and fragmentation are influenced by the temporal duration of the laser pulses, the intensity in the laser spot and the wavelength of the pulsed laser beam. Spontaneous fragmentations primarily occur only at high energy densities in the first nanosecond, for example; in contrast, most metastable ions are produced by irradiations with durations of longer than three nanoseconds.
Nowadays, commercially available MALDI mass spectrometers are predominantly equipped with pulsed laser systems in the ultraviolet spectral range (UV). Due to its limited lifetime, the low-cost nitrogen laser, with a wavelength of λ=337 nm, has mostly been replaced by frequency-tripled Nd:YAG lasers, with a wavelength of λ=355 nm, in high-quality MALDI mass spectrometers. The Nd:YAG laser is based on a YAG crystal (yttrium-aluminum-garnet: Y3Al5O12) doped with neodymium ions. In the Nd:YAG laser, the frequency of the strongest laser line, which appears at a wavelength of 1064 nanometers, is first doubled to the second harmonic frequency in a first nonlinear optical conversion crystal, producing green light, and then converted into the stated UV wavelength of the third harmonic frequency in a second nonlinear conversion crystal by mixing fundamental wavelength and second harmonic frequency. So-called phase matching must be fulfilled in both crystals, which is achieved by precise temperature control of the crystals to better than 0.1 degree Kelvin. For this purpose, each of the crystals is enclosed in an appropriately controlled oven. As is usual, the second conversion crystal is arranged in such a way that it compensates for the walk-off of the green light with respect to the fundamental wavelength in the first nonlinear conversion crystal as far as possible. The duration of the laser pulses used in the MALDI method is typically between 3 and 8 nanoseconds in the UV.
When the nitrogen lasers were replaced with Nd:YAG lasers, the degree of ionization of the analyte molecules surprisingly dropped dramatically, which initially could not be explained. Investigations by the applicant showed that the lower degree of ionization was connected with the transition from an erratic beam structure which is constantly changing over time in the beam profile of the nitrogen laser to an unmodulated and constant Gaussian profile of the Nd:YAG laser. The spatially and temporally modulated beam structure of the nitrogen laser was generated by lightning-like discharges in the nitrogen gas imaged onto the sample, and the form and position of these discharges changed from shot to shot. The solid-state laser, in contrast, delivered a circular beam with a Gaussian profile. The ion yield from a Gaussian profile spot with a diameter of around 100 micrometers was extremely low and had to be compensated for by increasing the energy density; but this caused a deterioration in the mass resolution and an enormous increase in sample consumption, partly because liquefied matrix material was splashed away during the desorption process. It was shown that a spot diameter of only five to ten micrometers improved the degree of ionization; but, kept below the fragmentation limit for the energy density, it supplied too few ions per laser shot. Suitable measures were therefore employed to generate a structured beam profile, which was imaged onto the sample as a spot pattern with around ten intensity peaks, each roughly 6 to 10 micrometers in diameter. This pattern generation led to a dramatic improvement. It was possible to achieve an increase in the degree of ionization of analyte molecules by a factor of 100 without saturation of the ion detector system and with extremely low sample consumption. The structured beam profile for the spot pattern can be generated by many means, such as the introduction of phase disturbances for transversely coherent beams (using crumpled plastic films, for example). The most uniform spot patterns are generated by commercially available lens arrays made of silica glass. The method of beam generation and the corresponding laser systems have been described in U.S. Pat. No. 7,235,781, which is hereby incorporated by reference.
When generating the spot pattern, it is important that all the individual spots have as nearly as possible the same energy density. If, for example, a square lens array made up of nine lenses is irradiated by a laser beam with Gaussian profile, the central lens will produce a spot with higher energy density due to the maximum in the Gaussian profile. If the energy density is increased here by 50 percent, the degree of ionization increases by more than a factor of 10, which makes the other spots of the pattern insignificant for the production of ions. If all the spots are to have approximately the same ionization, only a small, central part of the Gaussian profile of a greatly expanded laser beam can be used for the illumination of the lens array. The light in the remaining part of the Gaussian profile, by far the largest part of the painstakingly generated UV light, must be destroyed with the aid of diaphragms or other measures. Furthermore, the adjustment of the laser beam transverse to the pattern generator is very demanding.
When commercial MALDI time-of-flight mass spectrometers were in their infancy, laser pulse rates of 20 to 50 Hz were used because the nitrogen lasers could not be operated with higher pulse rates without dramatically reducing the number of shots during their life. A good mass spectrum is comprised of a several hundred to a thousand individual spectra; the acquisition of a good, low-noise mass spectrum from a thousand individual spectra thus took around 20 seconds. The switch to solid-state lasers soon allowed pulse rates of 1000 hertz and acquisition times of around one second. With the introduction of imaging mass spectrometry on thin tissue sections with several tens of thousands of pixels and a summed-up mass spectrum for each pixel, the desire for higher acquisition rates was soon voiced, but this requires laser systems with far greater power. In principle, the limit for the laser pulse rate in time-of-flight mass spectrometers is around 10 kilohertz because the 100 microseconds available for the acquisition of a single flight time spectrum are just about sufficient. In order to achieve laser pulse rates of 10 kilohertz, it is advisable to be careful with the laser energy and not destroy most of the UV light produced, because otherwise the high power demands would make the laser systems far too expensive and complex.
There is a need for a mass spectrometer with a low costs laser system for the ionization of a sample which can be operated with particularly high pulse rate, allows the ionization yield to be increased, has a long lifetime and can be operated with low energy consumption.